In vitro activity: Although displaying little impact on cell proliferation, Thiazovivin treatment significantly enhances the survival of human embryonic stem cells (hESCs) after enzymatic dissociation more than 30-fold, while homogenously maintaining pluripotency with the characteristic colony morphology, expression of typical pluripotency markers such as alkaline phosphatase (ALP), and normal karyotype. Dissociated hESCs treated with Thiazovivin display dramatically increased adhesion to matrigel- or laminin-coated plates but not to gelatin-coated plates within a few hours. Thiazovivin treatment increases cell-ECM adhesion-mediated β1 integrin activity, which synergizes with growth factors to promote cell survival. In addition to activating integrin, Thiazovivin but not Tyrintegin (Ptn) protects hESCs from death in the absence of ECM in suspension through E-cadherin-mediated cell-cell interaction. Thiazovivin treatment potently inhibits endocytosis of E-cadherin, consequently stabilizing E-cadherin on the cell surface and leading to reestablishment of cell-cell interaction, which is essential for hESC survival in ECM-free conditions. Thiazovivin but not Tyrintegin (Ptn) at 2 μM inhibits Rho-associated kinase (ROCK) activity and protects hESCs at a similar level as the widely used selective ROCK inhibitor Y-27632 at 10 μM, suggesting that Rho-ROCK signaling regulates cell-ECM and cell-cell adhesion. Thiazovivin at 1 μM increases the reprogramming efficiency of CB mononuclear cells to induced pluripotent stem cells (iPSCs) by more than 10 times.

Kinase Assay: Thiazovivin (2 μM) inhibits ROCK activity and protects human embryonic stem cells (hESCs).

Cell Assay: For cell proliferation assays, embryo-derived stem-like cells (eSLCs) are newly passaged and cultured in 3i system for 48 h on the feeder-free condition to prevent contamination of the BrdU-positive feeder cells. Cells are fixed with 4% paraformaldehyde in PBS (pH 7.4) at 37°C for 2 h, acid-treated with 2 N HCl in PBS for 30 min at 45°C, equilibrated with 0.1 M borate buffer (pH 8.5), and finally incubated with blocking buffer (20% Calf serum; 0.1% Triton X-100; 1% DMSO in PBS) for 2 h. Fixed cells are immunostained with antibodies against anti-BrdU mouse monoclonal antibody IgG followed by incubation with the secondary antibodies FITC conjugated goat anti-mouse IgG. The treated cells are covered with slow-fade anti-fade with DAPI for nuclear staining and covered with a glass coverslip. Images are captured with the fluorescence microscope.